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Journal of Virology, August 1999, p. 6293-6298, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Second-Site Reversion of a Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant That Restores Enzyme Function and Replication Capacity

Isabel Olivares,1 Víctor Sánchez-Merino,1 Miguel A. Martínez,2 Esteban Domingo,3 Cecilio López-Galíndez,1 and Luis Menéndez-Arias3,*

Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, 28220 Majadahonda (Madrid),1 Fundación Irsi-Caixa, Hospital Universitario Germans Trias i Pujol, Badalona (Barcelona),2 and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,3 Spain

Received 17 March 1999/Accepted 10 May 1999

Nonconservative substitutions for Tyr-115 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) lead to enzymes displaying lower affinity for deoxynucleoside triphosphates (dNTPs) (A. M. Martín-Hernández, E. Domingo, and L. Menéndez-Arias, EMBO J. 15:4434-4442, 1996). Several mutations at this position (Y115W, Y115L, Y115A, and Y115D) were introduced in an infectious HIV-1 clone, and the replicative capacity of the mutant viruses was monitored. Y115W was the only mutant able to replicate in MT-4 cells, albeit very poorly. Nucleotide sequence analysis of the progeny virus recovered from supernatants of four independent transfection experiments showed that the Y115W mutation was maintained. However, in all cases an additional substitution in the primer grip of the RT (M230I) emerged when the virus increased its replication capacity. Using recombinant HIV-1 RT, we demonstrate that M230I mitigates the polymerase activity defect of the Y115W mutant, by increasing the dNTP binding affinity of the enzyme. The second-site suppressor effects observed were mediated by mutations in the 66-kDa subunit of the RT, as demonstrated with chimeric heterodimers. Examination of available crystal structures of HIV-1 RT suggests a possible mechanism for restoration of enzyme activity by the second-site revertant.

* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-3978477. Fax: 34-91-3974799. E-mail: LMENENDEZ{at}CBM.UAM.ES.

Journal of Virology, August 1999, p. 6293-6298, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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